A reliable vitro semi-isolated preparation of the larval heart of Manduca sexta was developed to investigate the control of heartrate in the insect. Pharmacologically, the preparation was found to be sensitive only to the two biogenic amines, 5-hydroxytryptamine (5-HT, serotonin) and octopamine. Thresholds were 5 pmol and 50 pmol respectively. A typical response had both positive chronotropic and inotropic elements. The serotonin-induced response could only be mimicked by a limited range of less potent, structurally similar agonists, and could not be blocked by a number of recognised antagonists. The beta-blocker phentolamine failed to prevent the normal octopamine response. Inferences as to the nature of the receptor(s) were drawn. Endogenous cardioactive substances were found to be distributed in all neural tissues examined, but were concentrated in the abdominal nervous system. Gel-filtration chromatography of crude extracts revealed two bands of activity - a larger one of mw approximately 4000 and a second of less than 1000 daltons. Various techniques suggested the smaller factor was a small, heat-stable peptide, insensitive to trypsin and chymotrypsin, but whose activity was abolished by a bacterial protease and by performic acid oxidation. High performance liquid chromatography yielded partially purified material. The HPLC-purified fraction was found to act on the isolated larval hindgut and foregut of M. sexta and also on the hearts of other Lepidopteran species, causing increased frequency of contractions. In vivo recordings were rnade of heartrate during fourth and fifth larval and pupal stages. Injection of the partially purified cardioactive factor resulted in immediate cardioacceleration. A chromatographically similar factor was shown to be present in other stages of M. sexta and the release of this factor into the haemolymph during adult wing spreading was demonstrated. The thesis includes an overview of the pattern of the heartbeat in insects and a review of the evidence for cardioregulation by other invertebrate peptides.
|Date of Award||1984|